The invention relates to cell biology and oncology. More specifically, this invention relates to the methods for visualizing cellular organelles (such as a centrosome) and/or cytoskeletons (such as microtubules) through the use of crystallizing agents (such as tetrazolium salts), to a kit containing crystallizing agents adapted for such use, and to methods particularly for detecting neoplastic cells in a tissue sample, suspension, or a fluid sample by examining the cells for abnormalities in the size, number and shape of cellular organelles (such as a centrosome) and/or cytoskeletons (such as microtubules).
The characteristics and functions of cells are determined and maintained by cellular organelles and the cellular cytoskeleton. Cellular organelles include, but are not limited to, nucleus, mitochondria, peroxisomes, Golgi apparatus, lysosomes, endoplasmic reticulum, centrosome, and vacuoles. The term cytoskeleton (cytoskeletal structures) refers to an extensive scaffolding of fibrillar elements, including the three filamentous systems: microfilaments (or actin filaments), microtubules, and intermediate filaments. It may also include linin filaments. The components of the cytoskeleton are involved in diverse cellular functions ranging from mitosis to cell motility to signal transduction. Among these organelles and cytoskeletal structures, centrosome, microtubules, mitochondrion, endoplasmic reticulum, lysosomes, and nuclear envelope are most important.
The centrosome, a central body (or the major microtubule-organizing center (MTOC) of the cell) plays a key role in the temporal and spatial distribution of the interphasic and mitotic microtubule network. Therefore, the centrosome could be considered a major determinant of the overall organization of the cytoplasm and of the fidelity of cell division (Hsu, L. C. and White, R. L. (1998) Proc Natl Acad Sci U S A 27;95(22):12983-8). Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division, once and only once in each cell cycle, are all ensured through the actions of tightly regulated centrosomal function (Tanaka, T., et al., (1999) Cancer Res 59(9): 2041-4). Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis, and determines the number, polarity, and organization of interphase and mitotic microtubules (Tanaka, T., et al., (1999) Cancer Res 59(9): 2041-4; Pihan, G. A., et al., (1998) Cancer Res 58(17):3974-85). The main function of the centrosome is the nucleation of microtubules, and the controlled cycle of its duplication, the two duplicated entities functioning as mitotic spindle poles during subsequent cell division. Centrosomes and their associated microtubules direct events during mitosis and control the organization of animal cell structures and movement during interphase. Although the precise mechanisms by which duplicated chromosomes are equally segregated during mitosis are largely unknown, the centrosome is believed to play an important role(s) in the formation of bipolar spindles (Tanaka, T., et al., (1999) Cancer Res 58(17):3974-85). The microtubule nucleating ability of centrosomes of tissue sections is retained even after several years of storage as frozen tissue blocks (Salisbury, J. L., et al., (1999) J. Histochem. Cytochem. 47(10):1265-74).
In animal cells, the centrosome is composed of two centrioles surrounded by the so-called pericentriolar material (PCM), which consists of a complex thin filament network and two sets of appendages.
Malignant tumors generally display abnormal centrosome profiles, characterized by an increase in size and number of centrosomes, by their irregular distribution, abnormal structure, aberrant protein phosphorylation, and by increased microtubule nucleating capacity in comparison to centrosomes of normal tissues (Lingle, W. L. et al., (1998) Proc Natl Acad Sci U S A 95(6): 2950-5; Xu, X., et al., (1999) Mol Cell 3(3):389-95; Brinkley, B. R., et al., (1998) Cell Motil Cytoskeleton 41(4):281-8; Doxsey, S. (1998) Nat Genet 20(2):104-6; Kuo, K. K., et al., (2000) Hepatology 31(1):59-64). Among the abnormalities, centrosome hyperamplification is found to be more frequent in a variety of tumor types (Carroll, P. E., et al., (1999) Oncogene 18;18(11):1935-44; Hinchcliffe, E. H., et al., (1999) Science 283(5403):851-4; Xu, X., et al., (1999) Mol Cell 3(3):389-95).
Centrosome consists of many key proteins such as, SKP1p, cyclin-dependent kinase 2-cyclin E (Cdk2-E) (Hinchcliffe, E. H., et al., (1999) Science 283(5403): 851-4), kendrin (Flory, M. R., et al., (2000) Proc Natl Acad Sci U S A 23;97(11):5919-23), Protein kinase C-theta (Passalacqua, M., et al., (1999) Biochem J 337(Pt 1): 113-8), EB1 protein. Recently, a variety of cell cycle-regulated kinases or tumor suppressor genes are located in or are core components of the centrosome. They include Nek2 (Fry, A. M., et al., (1999) J Biol Chem 274(23): 1304-10), protein kinase A type II isozymes (Keryer, G., et al., (1999) Exp Cell Res 249(1):131-146), heat shock Cognate 70 (HSC70) (Bakkenist, C. J., et al., (1999) Cancer Res 59(17):4219-21), PH33 (Nakadai, T., et al., (1999) J Cell Sci 112 (Pt9):1353-64), AIKs (Kimura, M., et al., (1999) J Biol Chem 274(11)7334-40), human SCF(SKP2) subunit p19(SKP1) (Gstaiger, M., et al., (1999) Exp Cell Res 247(2)554-62), STK15/BTAK (Zhou, H., et al., (1998) Nat Genet 20(2): 189-93), C-Nap1 (Fry, A. M., et al., (1998) J Cell Biol 274(23): 1304-10), Tau-like proteins (Cross, D., et al., (1996) Exp Cell Res 229(2):378-87), cyclin E (Carroll, P. E., et al., (1999; Mussman, J. G., et al., (2000) Oncogene 23;19(13):1635-46), p53, retinoblastoma protein pRB and BRCA1(Hsu, L. C., et al., (1998) Proc Natl Acad Sci U S A 95(22):12983-8). These proteins are required in the initiation of DNA replication and mitotic progression (Gstaiger, M., et al., (1999) Exp Cell Res 15;247(2):554-62).
Microtubules, a filamentous system, are linear polymers of alpha- and beta (the beta1, beta2, and beta4 isotypes)-tubulin heterodimers. Except for being a frame of cellular membrane and organelles, microtubules may play an important role in other aspects. Microtubules are involved in diverse cellular functions ranging from mitosis to cell motility to signal transduction. Microtubules are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis (Shan, B., et al., (1999) Proc Natl Acad Sci U S A 96(10):5686-5691). They are nucleated by centrosome through the kinetochores of the centrosome. The spindle is a microtubule-based superstructure that assembles during mitosis to separate replicated DNA. Chromosome attachment to and movement on the spindle is intimately tied to the dynamics of microtubule polymerization and depolymerization. The sister chromatid pairs must maintain a stable attachment to spindle microtubules as the microtubules interconvert between growing and shrinking states. Drugs that are currently used in cancer therapy were designed to perturb microtubule shortening (depolymerization) or lengthening (polymerization) (Compton, D. A., et al., (1999) Science 286:913-914).
Other cytoskeletons such as membrane skeleton, microvilli, cilia, flagella, microfilaments, actin filaments, contractile ring, and intermediate filaments are all important in the organization of the cytoplasm and of the fidelity of cell division.
In addition to the centrosome and microtubules, other cellular organelles or cellular sub organelles such as mitochondrion, chromosomes, chromatin, nuclei, nuclear matrix, nuclear lamina, core filaments, nuclear envelope (NEs), nuclear pore complexes (NPCs), nuclear membrane, centrioles, pericentriolar material (PCM), mitotic spindle, spindle pole bodies (SPBs), contractile rings, proteasomes, telomere, plasma membranes, Golgi complexes, Golgi apparatus, endoplasmic reticulum (ER), endosomes, peroxisomes, proteasomes, phagosomes, ribosomes, are all important in maintaining a cell""s life. Endoplasmic reticulum, e.g. is the site of synthesis and maturation of proteins.
Therefore, identification of a novel less-costing, simple, and effective method for the visualization of cellular organelles and/or cytoskeleton is indeed necessary in cell biology, cell cycle, signal transduction, development biology, and cancer research.
However, most available methods for the visualization of the centrosome and other important cellular organelles and/or cytoskeleton are based on the antigen-antibody reaction (Lingle, W. L. et al., (1998) Proc Natl Acad Sci U S A 95(6): 2950-5; Xu, X., et al., (1999) Mol Cell 3(3):389-95; Brinkley, B. R., et al., (1998) Cell Motil Cytoskeleton 41(4):281-8; Doxsey, S. (1998) Nat Genet 20(2):104-6; Carroll, P. E., et al., (1999) Oncogene 18;18(11):1935-44; Hinchcliffe, E. H., et al., (1999) Science 283(5403):851-4; Xu, X., et al., (1999) Mol Cell 3(3):389-95). These techniques have been proved to be very costly, poorly reproducible, time consuming, and requiring of very strict conditions. Particularly, these methods can not be used to demonstrate the dynamic states of cells.
It is against this background, this invention provides a biochemical method for visualizing cellular organelles and/or cytoskeletons, by treating tissues or cells with crystallizing agents. The crystallizing agents or compounds used in this invention are a variety of tetrazolium salts. The cell-mediated reduction of some tetrazolium salts has long been used as a cell number-counting method (Berridge, M V, and Tan, A S., (1993) Arch Biochem Biophys 303(2): 474-482; Bernas, T., et al., (1999) Biochim Biophys Acta 12;1451(1):73-81; Abe, K., and Saito, H., (1999) Brain Res 29;830(1):146-54; Liu, Y., et al., (1997). J Neurochem 69(2):581-93; Abe, K., and Saito, H., (1998) Neuroscience Res 31: 295-305). In the visualization of cellular organelles and/or cytoskeletons, the application of tetrazolium salts has never been mentioned. The inventor of this invention has found that tetrazolium salts can specifically concentrate on cellular organelles and/or cytoskeletons of a variety of cells and tissues, with the formation of visible crystals in these places. The visualization of cellular organelles and/or cytoskeletons using a biochemical approach instead of the complicated immune methods provides a less costly, very simple, quick, and effective method for the visualization of cellular organelles and/or cytoskeleton. It provides a tool with great potential in studying cell biology, structural biology, cell cycle, signal transduction, development biology, and oncology.
The inventor has found that enzymes, such as dehydrogenases, are highly expressed in various tissues, and cell lines, particularly in cancerous cells. Except for mitochondrion, this enzyme is widely expressed in other structures of the cells such as, but not limited to, centrosomes, microtubules, endoplasmic reticulum, flagella, nuclear envelopes, lysosomes and other structures as mentioned above. When crystallizing agents, such as tetrazolium salts, are present, cellular enzymes, mainly succinate dehydrogenase (SDH), which is usually anchored on the cellular structures, will reduce the tetrazolium salts into related crystals that can be visualized under proper conditions. Additionally, the formation of crystals may act as a restraint to the separation of the duplicable organelles and to the movement of cytoskeletons.
Based on this discovery, the present invention features a method for visualizing cellular organelles and/or cytoskeletons, in tissue or in cell suspension, by treating tissue or cells with one or more crystallizing agents. The method includes obtaining a tissue sample or a fluid sample containing multiple cells from a variety of sources such as, but not limited to mammalian, microorganisms, and cancers; then fixing the tissue sample or cultured cell sample; contacting the tissue sample or cell sample with a crystallizing agent under conditions that permit the formation of visible crystals through the reduction of said crystallizing agent by the enzymes located in said cellular organelles and/or cytoskeletons in said cells or tissues; and then visualizing cellular organelles and/or cytoskeletons having crystals formed on them. The method allows for easy and quick visualization of cellular organelles and/or cytoskeletons that are usually observed through complex immunofluorescence staining or under electronic microscope.
Due to the high expression of the dehydrogenases in cancer cells, this invention is particularly useful in detecting the changes of the number and shapes of the neoplasm in their cellular organelles and/or cytoskeletons, thereby detecting neoplastic disease in the tissue or differentiating neoplastic cells from normal cells.
Since the dehydrogenating reaction usually takes place in the viable cells, this invention is particularly useful in monitoring the changes of the number and shapes of the cellular organelles and/or cytoskeletons of the cells at different time points. Therefore, this invention is helpful for better understanding of the regulating mechanisms in cell cycle and signal transduction, particularly for screening the drugs that specifically target cellular organelles and/or cytoskeletons of the cells for their actions.
The enzymes of this invention can be any protein(s) present in cytoskeletons and/or cellular organelles such as membrane skeleton, microvilli, cilia, flagella, microfilaments, actin filaments, contractile ring, microtubules and intermediate filaments or proteins present in cell organelles or cellular sub organelles such as centrosome, centrioles, pericentriolar material (PCM), mitotic spindle, spindle pole bodies (SPBs), mitochondrion, chromosomes, chromatin, nuclei, nuclear matrix, nuclear lamina, core filaments, nuclear envelope (NEs), nuclear pore complexes (NPCs), nuclear membrane, contractile rings, lysosomes, telomere, plasma membranes, Golgi complexes, Golgi apparatus, endoplasmic reticulum (ER), endosomes, peroxisomes, proteasomes, phagosomes, ribosomes. The enzyme of this invention can be a component of these organelles and/or cytoskeletons or be stored in these organelles and/or cytoskeletons. The enzymes of this invention can be a dehydrogenase such as alcohol dehydrogenase, beta-hydroxysteroid dehydrogenase, inosine monophosphate dehydrogenase, glucose alpha.-dehydrogenase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glycerol-3 phosphate dehydrogenase (mGPDH), glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, 3-.alpha.-hydroxysteroid dehydrogenase, lactate dehydrogenase, L-glutamate dehydrogenase, leucine dehydrogenase, aldehyde dehydrogenase, sarcosine dehydrogenase, amine dehydrogenase, telomerase, choline dehydrogenase, fructose dehydrogenase, succinate dehydrogenase, and sorbitol dehydrogenase. The enzyme can also be other proteins such as, but not limited to, pericentrin, cp140, centrin, .gamma.-tubulin, .alpha.-tubulin, .beta.-tubulin (U.S. Pat. No. 5,972,626), SKP1p, cyclin-dependent kinase 2-cyclin E (Cdk2-E), Protein kinase C-theta, EB1 protein, Nek2, protein kinase A type II isozymes, heat shock Cognate 70 (HSC70), PH33, AIKs, human SCF(SKP2) subunit p19(SKP1), STK15/BTAK, C-Nap1, Tau-like proteins, p53, retinoblastoma protein pRB and BRCA1. The enzyme of this invention may be other oxidation reductases. The enzyme of present invention is most likely to be succinate dehydrogenase.
The crystallizing agents of this invention may be any compounds that can be reduced by cellular enzymes located in the organelles or cytoskeletons, with the formation of visible crystals. It is preferably of tetrazolium compounds. Tetrazolium compounds refer to the compounds that contain tetrazole, tetrazolyl, tetrazolo-, or tetrazyl, and tetrazotic acids. Tetrazolium compounds may also be tetrazotized compounds. Example of crystallizing agents is pABT (p-Anisyl Blue Tetrazolium Chloride); pApNBT, p-Anisyl-p-Nitro Blue Tetrazolium Chloride; BSPT, Thiazolyl blue (2-2xe2x80x2-Benzothiazolyl-5-styryl-3-(4xe2x80x2-phthalhydrazidyl) tetrazolium chloride); BT, Blue tetrazolium chloride; BTSPT, 2-(2xe2x80x2-Benzothiazolyl)-5-styryl-3-(4xe2x80x2-phthalhydrazidyl)-tetrazolium chloride; CTC, (5-Cyano-2,3-ditolyl tetrazolium chloride); DMDPT, [3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium Bromide, 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; DSNBT, Distyryl nitroblue tetrazolium chloride; (1H)-tetrazole; IDNTT, Iodonitrotetrazoilum chloride; INT, Iodo Nitro Tetrazolium Violet Chloride, p-iodo nitrotetrazolium violet (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride; INpT, 2-(p-iodophenyl)-p-nitrophenyl-5-phenyltetrazolium chloride; mNBT, m-Nitro Blue Tetrazolium Chloride; mNNT, m-Nitro Neotetrazolium Chloride; MNSTC, 2,2-bis(2-methoxyl-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt; MTT, tetrazolium bromide, thiazolyl blue tetrazolium bromide, (3xe2x86x924,5-dimethylthiazol-2-yl!-2,5-diphenyltetrazolium bromide); NBMT, Nitro Blue Monotetrazolium Chloride; NBT, p-Nitro Blue Tetrazolium Chloride, Nitro blue tetrazolium chloride (2,2xe2x80x2-di-nitrophenyl-5,5xe2x80x2-diphenyl-3,3xe2x80x2-(3,3xe2x80x2-dimethoxy-4,4xe2x80x2-diphenylene)ditetrazolium chloride); NT, Neotetrazolium chloride (2,2xe2x80x2,5,5xe2x80x2-Tetraphenyl-3,3xe2x80x2(p-diphenylene)-ditetrazolium chloride; NTV, Nitrotetrazolium Violet; Thiazolyl blue; TB, tetrazolium blue chloride (3,3xe2x80x2xe2x86x923,3xe2x80x2-dimethoxy(1,1xe2x80x2-biphenyl)-4,4xe2x80x2-diyl]-bis(2,5-diphenyl-2H-tetrazolium)dichloride); NBT, Nitroblue tetrazolium chloride; oTTR, o-Tolyl Tetrazolium Red; PCTMB, sodium 3xe2x80x2-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate; PNBT, p-Nitro Blue Tetrazolium Chloride; PTB, Piperonyl Tetrazolium Blue; pTTR, p-Tolyl Tetrazolium Red; TC-NBT, Thiocarbamyl nitro blue tetrazolium chloride (2,2xe2x80x2-di-p-nitrophenyl-5,5xe2x80x2-di-p-thiocarbamylphenyl-3,3xe2x80x2[3,3xe2x80x2-dimethoxy-4,4xe2x80x2-biphenylene]-ditetrazolium chloride; TNBT, Tetranitroblue tetrazolium chloride; TPTT, 1,3,5-triphenyltetrazolium; TR, TTC, TPT, Tetrazolium Red (2,3,5-triphenyltetrazolium chloride); TV, Tetrazolium violet, Violet Tetrazolium, 2,3,5-Triphenyl-2-H-tetrazolium chloride, 2,5-diphenyl-3-[.alpha.-naphthyl]-tetrazolium chloride, 2,5-diphenyl-3-[1-naphthyl]-2H-tetrazolium chloride; VTB, Veratryl tetrazolium blue; WST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; XTT, 2,2-bis(2-methoxyl-4-notro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.
The crystallizing agents of this invention are materials that can be reduced by cellular enzymes located in the organelles or cytoskeletons, with the formation of visible crystals. Among the agents tested, tetrazolium salts are most effective. Cellular enzymes, mainly SDH, which is usually anchored on the cellular structures, will reduce the tetrazolium salts into related crystals that can be visualized under proper conditions. The visible crystals are formed where the said enzymes are located, therefore providing a method for viewing the enzyme-hosting cellular structures or components. In addition, the inventor has also found that tetrazolium compounds have differential targeting structures. For example, TV forms crystals in centrosome, therefore specifically crystallizing centrosomes; MTT forms crystals in microtubules, mitochondrion, and nuclear envelopes or endoplasmic reticulum; and TR forms crystals in envelopes and/or other structures close to envelopes such as endoplasmic reticulum. Therefore different tetrazolium salts or analogues may specifically crystallize certain cellular organelles or sub-organelles. It also suggested that SDH may have different forms or be located widespread in cells.
The tissue sample or cell sample can be from any source, such as, but not limited to mammalian, microorganism, vegetable, or parasite. The tissue sample or cell sample of this invention may be normal or neoplastic, from any part of a mammalian e.g., brain, neck, breast, lung, esophagus, liver, stomach, kidney, colon, rectum, skin, connective tissue, lymph node, blood vessel, nerve, ovary, bladder, uterus, testis, and bone. The tissue sample can be fresh, e.g., a biopsy sample, or can be from an archived sample, e.g., a frozen sample or a sample embedded in paraffin. The cell sample can from fluid, tissue cavity, tissue homogenate, tissue lavage, biopsy, cell lines, bone marrow and blood. The cell sample can be fresh, e.g., blood or bone marrow sample, or can be from an archived sample, e.g., a frozen cell lines.
Cellular organelles and cytoskeletons can have difference appearances, depending on sample origins, concentration of the agents used, and the duration of the contacting time.
Samples can be processed using methods known in the art, which includes tissue isolation and dissociation to release individual cells. Cells can be spun onto coverslips (xe2x80x9ccytospunxe2x80x9d), fixed, and subjected to visualization under microscope. Cells are preferably visualized directly under microscope in culturing flasks.
Any crystallizing agent that can be reduced by cellular enzymes with the formation of visible crystals can be used in the invention. It includes any tetrazolium compound, not limited to those described above such as TR, MTT, and TV. Any new compounds that contain tetrazolium tetrazole, tetrazolyl, tetrazolo-, or tetrazyl will fall in the scope of this invention. New tetrazolium compounds further include tetrazotic acids and tetrazotized compounds.
Crystals formed by the reduction of said crystallizing agents can be detected using detection methods known in the art, e.g., microscope. Naked eyes can also tell the occurrence of the reaction by examining the change of the color in the culture medium. The method of the invention for visualizing cellular structures may be used with other techniques such as immunofluorescence, immunoperoxidase staining, flow cytometry, or Western blot hybridization.
The present invention is also directed to a kit containing one or more crystallizing agents, such as tetrazolium red, MTT, and tetrazolium violet. The kit may comprise a pharmaceutically acceptable carrier medium.
Additional objects, advantages and novel features of this invention shall be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentality, combinations, and methods particularly pointed out in the appended claims.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will control. Unless otherwise indicated, materials, methods, and examples described herein are illustrative only and not intended to be limiting.
Various features and advantages of the invention will be apparent from the following detailed description and from the claims.